(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`15 December 2016 (15.12.2016)
`
`WIPOI PCT
`
`\9
`
`(10) International Publication Number
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`WO 2016/200645 A1
`
`(51)
`
`International Patent Classification:
`C07K 1/04 (2006.01)
`A61K 31/08 (2006.01)
`C07K1/06 (2006.01)
`A61K31/7084 (2006.01)
`A61K 47/48 (2006.01)
`A61K 38/27 (2006.01)
`
`(21)
`
`International Application Number:
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`PCT/USZOI6/O35111
`
`(22)
`
`International Filing Date:
`
`(25)
`
`(26)
`
`(30)
`
`(72)
`(71)
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`(81)
`
`Filing Language:
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`Publication Language:
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`31 May 2016 (31.05.2016)
`
`English
`
`English
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`Priority Data:
`62/174,528
`
`12 June 2015 (12.06.2015)
`
`US
`
`Inventor; and
`Applicant
`: WANG, Tianxin [US/US]; 510 Monarch
`Ridge Dr, Walnut Creek, California 94597 (US).
`
`Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`Ao, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, Fl, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
`MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM,
`
`PA, PE, PG, PH, PL, PT, QA, Ro, RS, RU, RW, SA, SC,
`SD, SE, SG, SK, SL, SM, ST, SV, SY, T11, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
`TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
`TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
`DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, KM, ML, MR, NE, SN, TD, TG).
`Declarations under Rule 4.17 :
`
`as to the identity ofthe inventor (Rule 4. l7(i))
`
`as to applicant’s entitlement to applyfor and be granted a
`patent (Rule 4.1 7(ii))
`
`as to the applicant’s entitlement to claim the priority ofthe
`earlier application (Rule 4.1 7(iii'))
`Published:
`
`with international search report (Art. 21(3))
`
`with sequence listing part ofdescription (Rule 5.2(a))
`
`(54) Title: METHODS FOR PROTEIN MODIFICATION IN PHARMACEUTICAL APPLICATIONS
`
`cleavable moiety
`$1thI)itor
`
`
`
`
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`Inactive enzyme
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`Fig. 21
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`
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`active enzyme
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`(57) Abstract: The current invention discloses methods to modify protein for pharmaceutical applications and reagents to treat dis -
`ease such as pathogen infection and cancer. The method involves increasing the molecular weight of the protein by connecting mul-
`tiple protein units with site specific conjugation to extend the in vivo half life. The current invention also discloses methods to con-
`struct affinity ligand in protein or aptamer form, which becomes active when they reach the treatment target, therefore provide higher
`specificity for treatment.
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`
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`WO 2016/200645
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`PCT/USZOl6/03511 1
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`Methods for protein modification in pharmaceutical applications
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`CROSS-REFERENCE TO RELATED APPLICATIONS
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`This application claims priority to U.S. Provisional Patent Application No. 62/174,528 filed on
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`June 12, 2015. The entire disclosure of the prior application is considered to be part of the
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`disclosure of the instant application and is hereby incorporated by reference.
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`BACKGROUND OF THE INVENTION
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`1O
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`Field of the Invention
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`The current invention relates to methods to modify protein for pharmaceutical applications and
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`reagents to treat disease such as pathogen infection and cancer. The current invention also
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`relates to methods to extend the in vivo half life and potency of protein and aptamer based
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`reagents.
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`Background Information
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`Protein drugs have changed the face of modern medicine, finding application in a variety of
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`different diseases such as cancer, anemia, and neutropenia. As with any drugs, however, the
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`need and desire for drugs having improved specificity and selectivity for their targets is of great
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`interest, especially in developing second generation of protein drugs having known targets to
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`which they bind. It is also desirable to have a long in vivo half life for the protein drug to reduce
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`their injection frequency to provide a better treatment for patient. Extending the half-life a
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`therapeutic agent, whether being a therapeutic protein, peptide or small molecule, often requires
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`specialized formulations or modifications
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`to the therapeutic agent
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`itself. Conventional
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`modification methods such as pegylation, adding to the therapeutic agent an antibody fragment
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`or an albumin molecule, suffer from a number of profound drawbacks. For example, PEGylated
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`proteins have been observed to cause renal
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`tubular vacuolation in animal models. Renally
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`cleared PEGylated proteins or their metabolites may accumulate in the kidney, causing
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`formation of PEG hydrates that interfere with normal glomerular filtration. Thus, there remains a
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`considerable need for alternative compositions and methods useful for the production of highly
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`pure form of therapeutic agents with extended half-life properties at a reasonable cost.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`FIG. 1 shows multivalent homo Fab format with suitable length flexible linker for higher affinity.
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`FIG. 2 shows hetero Fab format
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`targeting two antigens of the different protein on the
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`cell/microorganism for higher affinity.
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`FIG. 3 shows Hetero Fab format targeting two epitope sites of the same target protein for higher
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`affinity.
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`FIG. 4 shows construction of bi-specific antibody and ADC using selective reduction.
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`FIG. 5 shows bi specific antibody by linking two or more full size antibodies.
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`FIG. 6 shows an example of the preparation of bi specific antibody by linking two full size
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`antibodies.
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`FIG. 7 shows uses an example of using immobilized affinity group targeting the carbohydrate on
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`the antibody to selectively protect one FC conjugation site on the antibody to achieve mono
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`45
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`conjugation
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`FIG. 8 shows mono labeling of drug and linker on the antibody
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`FIG. 9 shows the structure and activating mechanism of self assembly probody
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`FIG. 10 shows examples of self assembly probody with Fc modifier
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`FIG. 11 shows the activation mechanism of self assembly probody with Fc modifier
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`FIG. 12 shows an example of self assembly probody with Fe modifier
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`FIG. 13 shows example of self assembly probody with heterogenic MM
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`FIG. 14 shows the structure and activating mechanism of protamer
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`FIG. 15 shows the structure and activating mechanism of self assembly protamer
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`FIG. 16 shows examples protamer with half life modifier or drug conjugation
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`FIG. 17 shows an example of Binding Based Prozyme, which is an enzyme activated upon
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`binding of aptamer
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`FIG. 18shows an example of Binding Based Prozyme, which is an enzyme activated upon
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`binding of antibody
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`FIG. 19 shows the scheme of ABP (antibody binding partner)—linker—E[P (enzyme inhibition
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`60
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`partner) based Prozyme
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`FIG. 20 shows the examples of format of ABP (antibody binding partner)—linker-E[P (enzyme
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`inhibition partner) based prozyme
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`FIG. 21 shows the scheme of Cleavage Based Prozyme, which is an enzyme activated with
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`second enzyme
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`65
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`FIG. 22 shows an examples of a block polymer made of two PEG blocks connected with a
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`biodegradable polylactic acid.
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`WO 2016/200645
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`PCT/USZOl6/035111
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`FIG. 23 shows different formats of biodegradable PEG and the biodegradable HGH dimer.
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`FIG. 24 shows an example of HGH trimer that can extend HGH in vivo halflife.
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`FIG. 25 shows an example of the HGH trimer and its preparation
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`7O
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`FIG. 26 shows an example of HGH trimer using 3 arm linker
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`FIG. 27 shows another example of HGH trimer using 3 arm linker
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`FIG. 28 shows the scheme of crosslink HGH with affinity group to extend its in vivo half life
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`FIG. 29 shows the scheme of crosslink HGH with antibody to extend its in vivo half life
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`FIG. 30 shows HGH trimer for half-life extension using a small PEG or peptide as linker and the
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`75
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`synthesis.
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`FIG.3l shows another example of HGH trimer for half-life extension using a small PEG as
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`linker and the synthesis.
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`FIG. 32 shows examples of HGH oligomer with biodegradable linker.
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`FIG. 33 shows an example of HGH oligomer with peptide linker prepared with recombinant
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`8O
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`technology.
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`FIG. 34 shows examples of HGH oligomer with terminal modifier.
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`DESCRIPTION OF THE INVENTIONS AND THE PREFERRED EMBODIMENT
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`The current invention discloses a method and formulation dosage form to improve the in vivo
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`half life and potency of biological active protein by combining protein with protein—antibody
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`immuno complex and administering it to the patient, in which the amount of protein is greater
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`than the binding capacity of antibody to provide free unbound protein in the formulation. In the
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`current inventions the “/” mark means either “and” or “or”.
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`90
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`The method comprises the following steps:
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`1) Administering protein-antibody immuno complex to the patient at the effective amount for
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`desired biological activity of the protein. This can be achieved by prepare the protein-antibody
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`immuno complex first and then administer it to the patient. Optionally the mixture of free
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`additional protein and the protein—antibody immuno complex can be used instead of protein—
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`antibody immuno complex only. This can also be done by administering protein and the
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`antibody separately to the patient to allow the formation of immuno complex in vivo. In some
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`embodiments the amount of the protein is equal or greater than the binding capacity of the
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`antibody. For example, if IgG is used, the amount of the protein is no less than two times of the
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`antibody amount (molar ratio) because each lgG binds with two proteins. In these embodiments
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`all the binding sites of antibody in the protein-antibody immuno complex are bound with protein.
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`Examples of suitable administering routes include intravenous, intraperitoneal, intramuscular
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`and subcutaneous routes and their combinations.
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`2) After a certain period of time when the in vivo concentration of the protein decreases to
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`undesired level, enough amount of protein is re-administered to the patient to maintain the
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`desired in vivo protein concentration (free and bound form). Sometimes additional protein—
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`antibody immuno complex can also be administered together with the protein to maintain the
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`desired in vivo antibody concentration, which results in desired protein-antibody immuno
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`complex concentration to ensure the sustained desired in vivo protein concentration.
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`3) Step 2 can be repeated several times based on the required in vivo protein concentration and
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`treatment length. For example, step 2 is repeated every 7 days or every 10 days or every two
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`weeks or every 20 days or every month for 3 months or 6 moths or 1 year or a few years.
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`The current invention discloses pharmaceutical formulation forms suitable for above method.
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`The pharmaceutical formulation form contains two or more dose, the first dose contains
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`effective amount of protein-antibody immuno complex or the mixture of free (unbound) protein
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`and the protein-antibody immuno complex. The second and later doses contain suitable amount
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`of protein drug only or the mixture of free protein and the protein-antibody immuno complex.
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`It is know that when antigen binds with antibody, the half life of the immuno complex can be
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`longer than that of the antigen alone, therefore provide longer in vivo half life, which is useful
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`for increase protein drug potency and reduce elimination. The antibody can also protect the
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`protein from enzyme degradation which also increase its half life and potency. However, the
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`dissociated protein has much faster clearance rate than the antibody therefore after the injection
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`ofimmuno complex, the ratio of protein vs antibody become smaller overtime and the
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`concentration of the protein decrease in a much greater extent than the decrease of antibody. The
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`unbound antibody will inhibit the protein activity, which further reduce the protein activity in
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`vivo. Repetitive injection of immuno complex will further increase the unbound antibody
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`concentration which will become an antibody trap therefore cannot provide satisfactory in vivo
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`protein activity for treatment. The current invention solves this problem by injecting free protein
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`only or mixture of free protein with the protein—antibody immuno complex, to maintain the
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`desired protein concentration without causing the buildup of antibody in vivo.
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`For example, a protein P (3OKD) is used for treating certain disease. Antibody lgG Abp (l 50KD)
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`is its neutralizing antibody. Using the method of the current invention, 6 mg of P is mixed with
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`15 mg Abp to prepare the immuno complex P-Abp in which each Abp binds with 2 P. At the
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`beginning of the treatment, 21 mg of P-Abp is injected (i.V.) to the patient. The in vivo half life
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`ofP is 10d and 20d for Abp and 70% Abp left on day 10 (based on the concentration of both
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`free form and bound form in immuno complex after administering P—Abp). The in vivo half life
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`of P not in immuno complex is 0.5 d. Therefore, a second dose containing 3~6mg of P is given
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`on day 10 and a third dose containing 10.5 mg of P-Abp and 3 mg ofP is given on day 20 to
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`maintain a steady effective in vivo protein P concentration. This can be repeated until the
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`treatment is finished (e.g. another second dose on day 30 and another third dose on day 40). The
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`formulation form will contain 1 first dose (21 mg of P-Abp) and multiple second doses (3~6mg
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`145
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`of P) and multiple third doses (mixture of 10.5 mg of P-Abp and 3 mg of P).
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`Alternatively, 21 mg of P—Abp is injected (i.V.) to the patient at the beginning of the treatment,
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`and a mixture of 3 mg ofP with 6.3mg Abp—P (3 0% of21mg because 30% of Abp is cleared on
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`day 10) is injected every 10 days. The formulation form will contain 1 first dose (21 mg of P—
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`Abp) and multiple second doses (mixture of 3 mg of P with 6.3mg P-Abp). 1f high dose of free P
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`does not cause adverse effect, 3X second doses can be injected on day 1 instead of21 mg P-Abp,
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`therefore the drug formulation only need to contain multiple of mixture of 3 mg of P with 6.3mg
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`P-Abp.
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`Besides what listed above, other scheme of the administering dose/interval and formulation
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`composition can be used to achieve the desired in vivo P concentration. The pharmacokinetics
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`(e. g. in vivo half life) can be measured for each individual to prepare the personalized medical
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`treatment. Averaged pharmacokinetics data from a large population can also be used instead to
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`design the composition of the formulation and administering schedule. Other route (e. g.
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`subcutaneous or intramuscular injection) can also be used.
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`There are many protein drugs can be used according the above method described in the current
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`invention. For example, HGH and antibody against HGH (either neutralizing IgG or non
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`neutralizing IgG, the best antibody can be obtained by screening), IL—7 and M25 antibody,
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`human IL—lO (hIL— 10) and humanized antihuman IL—lO ( hOLhIL—10) can be used for the current
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`application.
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`In some embodiments, the antibody-antigen protein drug complex used has a molar ratio of
`
`antibody : antigen > 0.5, which means some of the antibody binding sites do not bind with
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`antigen protein drug, to achieve a more steady blood drug concentration change. For example,
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`antibody bound with antigen at 1:1 ratio (half of the binding sites are empty in each antibody) is
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`used as antibody—antigen drug immuno complex. In one example, the first dose of is the
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`antibody bound with antigen drug at 1:1 ratio, the second and later dose contains two parts: free
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`antigen drug and antibody bound with antigen drug at 1:1 ratio. The two parts can be injected at
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`the same time or sequentially.
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`Example: Development plan for HGH affinity dosing
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`l) Antibody Screening:
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`-Mix several antibody (monoclonal from mouse, many commercially available) against HGH
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`180
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`with HGH and inject to the mouse
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`—Measure the serum HGH level and select the antibody that extend the HGH half—life the most
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`2) Dosing Screening :
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`-Adjust the ratio between Ab: HGH of the first dose to select the one providing the best PK
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`profile after the first dose
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`-Adjust the ratio between Ab: HGH of the later doses to select the one providing the best PK
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`profile ( or weight gain) during the later dose, the adjustment can be designed based on the pK
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`model developed during screening.
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`3) Humanizing: Antibody humanization and Dosing adjustment for human
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`The current invention also discloses novel strategy for site specific conjugation of proteins
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`including antibodies. Site specific antibody drug conjugation is a promising drug discovery
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`195
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`strategy for cancer treatment; several companies (e. g.
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`ambrx, innate-pharma and sutrobio) are
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`WO 2016/200645
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`working on developing new method for site specific conjugation of proteins, In one aspect, the
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`new method in the current invention uses elevated temperature for site specific conjugation
`
`using MTgase (microbial transglutaminase, also called bacterial transglutaminase, BTG) to
`
`couple the drug/linker having amine group to the Gln of the protein. Preferred temperature is >
`
`200
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`40 degree, more preferably > 45 degree but less than 75 degree. In some embodiments, the
`
`temperature is 50 ~ 65 0C. The elevated temperature can expose the previous hidden (e. g. the
`
`Gln in antibody difficult to be accessed by MTgase) functional groups for site specific
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`conjugation.
`
`In one example conjugation of IgGl with Monodansylcadaverine (MDC) is catalyzed by
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`205
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`MTgase. MDC has a primary amine and its fluorescence can be easily monitored. MDC is used
`
`here to conjugate to mAB. To purified IgGl (l-lOmg/ml) in Tris-buffer (pH 6.5-8.5), add lV[DC
`
`(Sigma-Aldrich) in DMSO to final concentrations of 1-5 mM (final DMSO 2—lO%). Add
`
`purified MTgase to a final concentration of 005-1 .0 mg/ml. Incubate the reaction mixtures at
`
`50°C for 5 hours. Reaction is monitored by HPLC. Antigen peptide for the IgG (e.g. 5 fold
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`210
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`excess) can be added to the reaction mix to stabilize the Fab of the antibody.
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`In another aspect, the new method in the current invention uses MTgase to couple the
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`drug/linker having Gln group to the amine group of the protein (6. g. lysine or N terminal amine).
`
`The coupling can be done in either high temperature (e. g. 45~55 0C ) or low temperature ( e. g.
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`25-37 0C). Point mutation can be used on the protein (e. g. antibody) to introduce lysine as
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`215
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`coupling site.
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`In one example, pegylation of IgGl with 1 kDa PEG-CO-Gln-COOH or PEG-CO-Gln-Gly-NH2
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`is performed by MTgase catalysis. This experiment is carried out essentially the same condition
`
`as described in the example above. The MDC is replaced with MW: 1 k PEG-CO-Gln-COOH
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`(the product of HO-PEG—COOH coupling with Gln, which for an amide bond between PEG-
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`220
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`COOH and the amine of Gln) or PEG—CO—Gln—Gly—NHZ in pH 7.0 to a final concentration of l
`
`to 2mM, PEGylated IgGl is obtained. The Gln of on the PEG couples to the amine group on the
`
`IgGl by MTgase catalysis.
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`The current invention also discloses novel toxin which can be used for antibody-drug conjugate
`
`(ADC) and cancer treatment. Currently MIVIAE (monomethyl auristatin E) or MMAF is used
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`225
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`for ADC as toxin to conjugate with antibody. The novel toxins in the current invention are N-
`
`substituted MMAE/MIVIAF . Their structures are shown below (the attachment group is where
`
`the toxin to be conjugated with):
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`

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`WO 2016/200645
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`PCT/USZOl6/03511 1
`
`§H
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`v.-
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`
`230 Where in R1, R2 and R3 is independently selected from the group consisting of H, C1-C8 alkyl,
`
`haloCl-C8 alkyl, C3-C8 carbocycle, aryl, X-aryl, OR21, SR21, N(R21)2, —NHCOR21 and —
`
`NHSOR2R21, X—(C3-C8 carbocycle), C3-C8 heterocycle and X—(C3-C8 heterocycle), each
`
`X is independently C1—C10alkylene.
`
`In some examples, R1 is independently H or CH3 or CH2F or CHF2 or CF3, R2 independently
`
`235
`
`is H or CH3 or CH2F or CF3 and R3 is independently H or CH3 or CH2F or CF3.
`
`The structures also include:
`
`.-'Tiff}.ffffffflflflffffffiTfylfffflflffffffiffffx
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`

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`WO 2016/200645
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`PCT/USZOl6/03511 1
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`
`
`Where in R1, R2 and R3 is independently selected from the group consisting of H, C1-C8 alkyl,
`
`240
`
`haloCl-C8 alkyl, C3-C8 carbocycle, aryl, X-aryl, OR21, SR21, N(R21)2, —NHCOR21 and —
`
`NHSOR2R21, X—(C3-C8 carbocycle), C3-C8 heterocycle and X—(C3-C8 heterocycle), each
`
`X is independently Cl—ClOalkylene, n is an integer between 1 ~5.
`
`245
`
`250
`
`In some examples, R1 is independently H or CH3 or CH2F or CHF2 or CF3, R2 independently
`
`is H or CH3 or CHZF or CF3 or isopropyl and R3 is independently H or CH3 or CH2F or CF3.
`
`The attachment group is where the toxin conjugates to linker or proteins. It is the same as those
`
`used in the current MMAE/MMAF ADC.
`
`The current invention also discloses novel strategy for antibody purification and conjugation.
`
`Current antibody purification method uses protein A column, which is expensive and has
`
`potential risk of leaking protein A. The new strategy uses affinity column based on epitope
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`peptide or mimotope for antibody purification by coupling epitope peptide or minotope to the
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`solid phase support as column filler, e. g. sephadex beads. The advantages are low cost, more
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`stable chemistry for immobilization, selectively isolating antibody with high binding affinity and
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`removing non binding antibody/ADC, therefore increase the potency and therapeutic index of
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`antibody or ADC. In one example: peptide NIYNCEPANPSEKNSPSTQYCYSI (SEQ ID NO: 1)
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`is used to couple to solid phase support to make an affinity column, which can be used for
`
`Rituximab purification. The benefit of using peptide based affinity column (activated beads are
`
`commercially available) is greater than the effort of developing the peptide for each antibody.
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`Many peptide sequence are available from literature or epitope scan for both linear and
`
`conformational discourteous epitope (e. g. from pepscan). This strategy also works for other
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`protein drugs by using synthetic ligand (e. g. affinity peptide) for the binding site of that protein
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`to prepare affinity column.
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`Furthermore, it can be used to selectively protect the reactive amino acid in the binding site of
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`the antibody, by adding epitope peptide or mimotope (free form or immobilized) or masking
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`peptide (e, g. those used in probody) to form the peptide-antibody complex during antibody-drug
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`conjugation. Similarly it can be used to protect the active binding site of other type of protein by
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`using the affinity ligand that can mask the active binding site of that protein. This method is
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`suitable for both chemical and enzymatic conjugation, therefore provide more drug load for
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`ADC, more conjugation reaction can be allowed (e. g. >2 types of toxin). Similar strategy is used
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`in enzyme conjugation to keep the enzyme activity by adding enzyme substrate. Synthetic
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`peptide is very easy to make (low cost and more stable) using synthetic peptide chemistry than
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`making proteins. Peptide can be made in large amount easily using solid phase peptide synthesis.
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`In one example: peptide NIYNCEPANPSEKNSPSTQYCYSI (SEQ ID NO: 1) is used to protect
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`Rituximab during conjugating drugs to the antibody. Peptide
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`NIYNCEPANPSEKNSPSTQYCYSI (SEQ ID NO: 1) can bind with Rituximab at its antigen
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`binding site. By adding NIYNCEPANPSEKNSPSTQYCYSI (preferably at > 2:1 ratio) to
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`Rituximab before chemical conjugation on Rituximab, the antigen binding site of Rituximab is
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`protected.
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`The current invention also discloses novel Bi specific antibody and its application. They can be
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`used to treat cancer, pathogens, immune disorders and targeting delivery of vector (retrovirus
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`based gene therapy).
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`Bi specific antibody can be in traditional monomer format: multivalent homo Fab format with a
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`suitable length flexible linker for higher affinity (not bi specific), hetero Fab format targeting
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`two epitope sites of the different protein on the cell/microorganism to achieve higher affinity
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`and hetero Fab format targeting two epitope sites of the target protein to achieve higher affinity.
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`Bi specific antibody can also be in dimer format or trimer or higher degree oligomer format:
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`multivalent homo Fab format with suitable length flexible linker for higher affinity (not bi
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`specific), hetero Fab format targeting two epitope sites of the target protein for higher affinity
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`and hetero Fab format targeting two epitope sites of the different protein on the
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`cell/microorganism for higher affinity. Construction of this type of Bi specific antibody can be
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`achieved using boric affinity column or lectin affinity column for mono conjugation (boric
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`affinity column or lectin affinity column can also be used for antibody purification).
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`Bi Specific Antibody (BsAb) can be used for against cytoplasm target. In some embodiments, Bi
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`specific antibody is in traditional antibody monomer format: multivalent homo Fab format with
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`suitable length flexible linker for higher affinity. Native antibody’s hinge region is not long and
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`flexible enough therefore may not reach two antigens on the target cell. Using a flexible and
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`suitable length of linker to connect the antibody parts will greatly increase the binding affinity
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`(figure 1). The linker can be a flexible peptide linker such as poly glycine/serine or synthetic
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`polymer such as PEG. In the current inventions the “/” mark means either “and” or “or”.
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`It can also be hetero Fab format targeting two antigens of the different protein on the
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`cell/microorganism for higher affinity. Similarly, the above approach can also be applied to
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`bispecific antibody binding to two different antigens on the cell/pathogen. The bispecific
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`antibodies with flexible proper length linkers can be made easily to get the optimal binding of
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`two antigens simultaneously while traditional method is time consuming (figure 2).
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`Another format is to use bi specific antibody to target the two different epitopes on the same
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`antigen, which will also significantly increase the binding affinity (figure 3).
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`Construction of these types of Bi specific antibody: Using the selective reduction of the disulfide
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`bond at the hinge region with 2-Mercaptoethylamine , several formats (figure 4) can be used to
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`make this type ofbispecific antibodies, with high yield and no concern for dimer formation to
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`ease the industrial scale separation process. Two formats are shown below: to use some —SH
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`reactive reagent (or mutation to remove -SH) to block the free —SH group to prevent the
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`regeneration of —SS- bond, which will generate the traditional format bispecific antibody.
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`Similarly, bi specific antibody by linking two or more full size antibodies can also be used in
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`above applications (figure 5) and formats and synthesized readily (figure 6), which may offer
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`higher stability and higher binding affinity as shown by IgA and IgM.
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`Construction of this type of Bi specific antibody can be achieved using borate affinity column or
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`lectin affinity column for mono conjugation. This strategy is also useful for antibody
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`purification. This design uses immobilized antibody to archive high yield mono labeling of the
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`antibody, to eliminate the potential bi—labeled antibody (generating polymerized antibody).
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`Immobilized protein was used to make mono PEGlated protein previously. Ion exchange resin
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`was used to immobilize the protein. However ion exchange resin may not work for antibody to
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`block half of FC and the binding affinity is low, which may cause exchange between two sides.
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`This design uses affinity group targeting the carbohydrate on the antibody to selectively protect
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`one FC conjugation site on the antibody to achieve the mono conjugation. Suitable affinity
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`resins include borate based affinity solid phase support or lectin based affinity phase support
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`(figure 7). When one side of the antibody is protected, the other side can be selectively modified
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`(e.g. site specific conjugation using enzyme such as mTGase).
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`Borate is a carbohydrate chelators and borate based column is widely used in separating
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`carbohydrate, many are commercially available (6. g. from Sigma). Different borate also has
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`different affinity to different sugar. Lectins are carbohydrate-binding proteins, most are from
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`plant, which is used as antivirus/bacterial drug for animals. Different lectin has selectivity for
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`different carbohydrate. Lectin column is also used in studying carbohydrate. Lectin or borate
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`based resin can also be a useful tool for large scale purification of antibody drugs during ADC
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`labeling. They can also be used for protein mono labeling other than antibody if the protein has
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`carbohydrate modification.
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`If mono labeling drug on the antibody can be done efficiently, then the later mono labeling of
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`linker labeling can be done easily (figure 8).
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`Using ADC made of BsAb against two makers on the target cell will increase the specificity of
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`drug delivery.
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`Bi Specific Antibody can be used for cytoplasm target. For example, in lupus, the key auto
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`antibody causing the damage to the cells is the auto antibody against dsDNA. They are released
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`from lysosome after internalization and bind with nucleus to cause cell damage. There are also
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`many antibodies are against cytoplasm target. It is known that many cell surface receptors are
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`reused after been internalized: suggesting it is not digested in lysosome.
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`Similarly, antibody against tublin can be used instead of MIVIAE or other toxin in the ADC.
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`Therefore the ADC is essentially an antibody (eg. for HER2)-antibody (e.g. for tubulin)
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`conjugate, in another word, a bi-specific antibody. The advantage of using antibody instead of
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`toxin as effector is that AB is much less toxic and can have high affinity and specificity,
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`therefore less concern on side effect and toxicity due to potential release of toxin in blood
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`circulation. Furthermore, the effector antibody may not need to target tubulin, it can be antibody
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`against many other cytoplasm in tumor cells (e. g.
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`tolemarase).
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`One issue with ADC for drug is that there are limited cell surface markers on cancers cells can
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`be used for antibody and even HERZ is only positive in 30% patients. To expand the application
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`of the above BS—Antibody strategy, the targets can be extended to diseases beyond cancer. There
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`are many cytoplasm targets for many diseases and a lot of drugs are against cytoplasm targets,
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`bi-specific antibody can be used as therapeutics against them: one AB against cytoplasm target
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`and one against cell surface marker to help the effector AB uptaken by the cell.
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`The rate of internalization of antibody dimer should not be a big problem as size is not a key
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`factor affecting internalization in many cases. A much bigger virus can be internalized easily.
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`Even if it was a concern, monomer type Bs antibody or adding a positively charged linker can be
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`used to improve internalization.
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`An antibody (against gp120) —toxin conjugate has been made to kill HIV virus infected T cell
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`(HIV infected T cells express HIV gp 120 on T cell surface). This strategy can be applied to
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`many other virus infections since the infected cell will express virus protein on their surface.
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`However, toxin is toxic and has their limitations.
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`A more universal strategy is to use antibody-virus inhibitor conjugates instead. Many virus
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`inhibitors are very potent and have suitable functional groups to be linked to antibody with very
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`low toxicity to cells. For example, antibody against gplZO or CD3, CD4 can be conjugated to
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`HIV RT inhibitor (e. g. AZT ) or HIV protease inhibitor(e. g. Amprenavir) to treat HIV infection;
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`antibody against CKlS, CKl9 or HBV surface antige